Make sure you know the direction of the horizontal axis of the
chromatogram (usually, either volume or time) - it may run from right to left
or vice versa - and make a note of the detector sensitivity on the vertical axis.
Ideally, the base line should be 'flat' between peaks, but it may drift up or
down owing to a number of factors including:
- changes in the composition of the mobile phase (e.g. in gradient elution);
- tailing of material from previous peaks;
- carry-over of material from previous samples; this can be avoided by
efficient cleaning of columns between runs - allow sufficient time for the
previous sample to pass through the column before you introduce the
- loss of the stationary phase from the column (column 'bleed'), caused by
extreme elution conditions;
- air bubbles (in liquid chromatography); if the buffers used in the mobile
phase are not effectively degassed, air bubbles may build up in the flow
cell of the detector, leading to a gradual upward drift of the base line,
followed by a sharp fall when the accumulated air is released. Small air
bubbles that do not become trapped may give spurious small peaks as
they pass through the detector.
Peak close to the origin may be due to non-retained sample molecules,
flowing at the same rate as the mobile phase, or to artefacts, e.g. air (GC) or
solvent (HPLC) in the sample. Whatever its origin, this peak can be used to
measure the void volume and dead time of the column. No peaks
from genuine sample components should appear before this type of peak.
Peaks can be denoted on the basis of their elution volume (used mainly in
liquid chromatography) or their retention times (mainly in GC). If the peaks
are not narrow and symmetrical, they may contain more than one component.
Where peaks are more curved on the trailing side compared with the leading
side, this may indicate too great an association between
the component and the stationary phase, or overloading of the column.